Glucose metabolism is of vital importance in maintaining proper synaptic transmission and brain function. Acute glycemia induces aberrant synaptic transmission and brain function without a significant reduction in global cellular ATP levels. The mechanism is not well understood at the molecular level. We have recently provided evidence that glyceraldehyde phosphate dehydrogenase and 3-phosphoglycerate kinase, glycolytic enzymes responsible for production of ATP, are associated with synaptic vesicles and that activation of these enzymes is sufficient to support glutamate uptake into synaptic vesicles. Additional evidence suggests that glycolytically and locally produced ATP on the surface of synaptic vesicles, rather than ATP synthesized in mitochondria, plays an important role in accumulating glutamate into synaptic vesicles in the nerve ending. More recently we have obtained preliminary evidence that pyruvate kinase (PK), another glycolytic enzyme capable of ATP synthesis, is also bound to synaptic vesicles, and that activation of this enzyme leads to marked glutamate uptake into synaptic vesicles. Glutamate is a major excitatory neurotransmitter in the vertebrate central nervous system, and proper glutamate transmission is essential for neuronal communication, learning and memory function. Of interest, phenylpyruvate, whose blood levels are high in phenylketonuria, was found to inhibit PK-mediated vesicular glutamate uptake, suggesting some clinical implication for this vesicle-bound enzyme. We postulate that the synaptic vesicle-bound glycolytic enzyme PK is functionally coupled to V-type proton pump ATPase, which generates an electrochemical proton gradient (the driving force for glutamate uptake into synaptic vesicles) and thereby plays a critical role in setting the stage for glutamate transmission. We plan to provide sufficient evidence to support the hypothesis mentioned above, with the following aims: (1) to characterize vesicle-bound PK-mediated glutamate uptake into synaptic vesicles, and (2) to demonstrate that PK and phenylpyruvate play important roles in vesicular accumulation of glutamate in synaptosomes. To achieve aim 1, we will study phosphoenol pyruvate plus ADP-dependent uptake in a number of respects, using isolated rat brain synaptic vesicles. To achieve 2, we will determine the effect of membrane-permeant PK inhibitors on vesicular glutamate content in and release from synaptosomes. This study is expected to contribute to better understanding of the relation between glucose metabolism and synaptic transmission/brain function. It is hoped that knowledge gained from this research will be of use in creating a new approach to treatment of certain neurological and mental disorders.